A role for glycosylated serine-rich repeat proteins in gram-positive bacterial pathogenesis

Abstract

Bacterial attachment to host surfaces is a pivotal event in the biological and infectious processes of both commensal and pathogenic bacteria, respectively. Serine-rich repeat proteins (SRRPs) are a family of adhesins in Gram-positive bacteria that mediate attachment to a variety of host and bacterial surfaces. As such, they contribute towards a wide-range of diseases including sub-acute bacterial endocarditis, community-acquired pneumonia, and meningitis. SRRPs are unique in that they are glycosylated, require a non-canonical Sec-translocase for transport, and are largely composed of a domain containing hundreds of alternating serine residues. These serine-rich repeats are thought to extend a unique non-repeat (NR) domain outward away from the bacterial surface to mediate adhesion. So far, NR domains have been determined to bind to sialic acid moieties, keratins, or other NR domains of a similar SRRP. This review summarizes how this important family of bacterial adhesins mediates bacterial attachment to host and bacterial cells, contributes to disease pathogenesis, and might be targeted for pharmacological intervention or used as novel protective vaccine antigens. This review also highlights recent structural findings on the NR domains of these proteins.

Figure 1. Structural domain organization of select Serine-rich repeat proteins (SRRPs)

SRRPs are composed of an N-terminal signal peptide (S); most often a single, but sometimes two, unique non-repeat domains (N) that mediate adhesion; two serine-rich repeat domains (SRR) flanking the single or second N domain (SRR1 and SRR2); and a C-terminal cell wall anchor domain (A). The SRRs are composed of serine residues alternating, most frequently, either an alanine, valine, or threonine residue. The large number of repeats in the SRR2 domain is responsible for the large size of most SRRPs. Domains are not drawn to scale.

Figure 2. Genetic organization of loci encoding SRRPs in Streptococcal and Staphylococcal species

SRRPs are encoded on genomic islands present in the chromosome. In addition to the SRRP (black arrow), these loci also contain numerous genes encoding enzymes responsible for the O-linked glycosylation of the protein (tan arrows) and for transport of the protein (blue arrows). No obvious regulatory elements are present. All SRRP loci have a core set of eight genes consisting of an SRRP and secY2, secA2, asp1-3 and gtfA-B (red dashed line).

Figure 3. SRRPs are glycosylated and extend the NR domains outward from the bacterial surface

A) Western blot and carbohydrate staining of whole cell lysates of a S. pneumoniae mutant deleted of its chromosomal PsrP but complemented with a plasmid (pfcsRK::PsrP_1–734) that expresses a truncated version of the protein with only 33 SRR repeats when grown in THY media supplemented with 1% fucose. Carbohydrate staining was done using a Periodic Acid-Schiff (PAS) stain. Note the presence of a faint fucose-inducible band at 80 kDa that is the predicted unglycosylated size of the recombinant protein (black triangle), and a stronger band at 210 kDa (white triangle). Only the 210 kDa band tested positive for carbohydrates by PAS, suggesting PsrP is glycosylated. Taken from (Shivshankar et al., 2009) B) Hypothetical model showing how SRRPs might employ the SRR2 domain to extend the NR domain outward away from the cell through surface components such that it can mediate adhesion. C) Immunogold electron microscopy using antibodies against the NR domain of PsrP. Note that the gold particles are found distal to the bacterial surface, supporting the model shown in panel B. Findings first described in (Shivshankar et al., 2009).

Figure 4. Aging enhances susceptibility to pneumococcal pneumonia in a K10-dependent Manner

A) Densitometric analyses and representative Western blot bands for K10 in human lung biopsies obtained from young (43–50 years; n = 6), mature (51–64 years; n = 8), and aged (64–89 years; n = 8) humans. Asterisks denote a statistical significant difference when using one-way ANOVA (Duncan’s Method; P < 0.05). B) Kaplan–Meier plot showing percent survival of aged Balb/c mice intranasally challenged with 10⁵ CFU of wild type S. pneumoniae (Wild type: n=8) or an isogenic mutant deficient in the SRRP PsrP (ΔpsrP: n=11). Statistical analysis was performed using a Kaplan–Meier log-rank Test. These findings where first described in (Shivshankar et al., 2011).

Figure 5. SRRPs expressed in medically relevant bacteria play a role in pathogenesis

PsrP in S. pneumoniae is an intra-species adhesin that initiates attachment to the host by binding to keratin 10 expressed on lung alveolar epithelium. Likewise, it can also mediate attachment to another PsrP molecule expressed on another pneumococci, facilitating bacterial aggregation, leading to pneumonia. GspB and Hsa are present on S. gordonii M99 and challis, respectively. These SRRPs bind to sialic acid moieties present on the platelet receptor GPIbα forming vegetative plaques causing infective endocarditis. SraP present in S. aureus mediates direct binding to platelets also forming vegetative plaques, and leading to infective endocarditis. Neonatal sepsis and meningitis is highly associated with the presence of Srr-2 in S. agalactiae. The receptor or ligand of Srr-2 has not been identified, but it is suggested that expression of Srr-2 accelerates disease onset in neonates by facilitating transmigration of the BBB. Periodontal disease is associated with the expression of Fap1 from S. parasanguinis. Fap1 binds to glycosylated salivary proteins forming biofilms to cause caries. SrpA in S. cristatus is required for the formation of corn-cob structures with Corynbacteriummatruchoiti and Fusobacteriumnulceatum, mediating intra-species adhesion seen in dental plaque.

Figure 6. PsrP in S. pneumoniae mediates robust biofilms in vitro and pneumococcal aggregates in vivo

Micrographs of A) TIGR4 and B) T4 ΔpsrP biofilms grown in a flow cell under once-through flow conditions for 3 days. Bacteria were visualized with Live/Dead BacLight stain using an inverted confocal laser scanning microscope at 400× magnification. Representative micrographs of C) TIGR4 and D) T4 ΔpsrP Gram-stained bacteria from bronchial alveolar lavage samples from mice. These findings were first described in (Sanchez et al., 2010).

Figure 7. Modular organization of the NR regions within the SRRP family

Subdomains of the NR region organized according to structural similarities. Similarities are based on crystal structure and predicted sequence identity after BLAST or Clustal W sequence alignments(Garnett et al., 2012, Pyburn et al., 2011, Chenna et al., 2003, Altschul et al., 1990). The middle column lists the known ligands for these NR. Figure adapted from Pyburn et al.(Pyburn et al., 2011).